The invention relates to flow cytometers and to methods of calibrating and using flow cytometers.
Flow cytometers are widely used for analysing properties of cells that are suspended in a liquid. The liquid is passed through a conduit so that the cells flow past a fluorescence-based sensing device one at a time. The cells can thus be counted and classified according to their spectroscopic properties which can then optionally be used to further direct the flow or for other purposes. For example, the cells can be sorted downstream of the detection device according to their fluorescence properties. This is usually referred to as fluorescence activated cell sorting (FACS).
The instrument typically is provided with multiple detectors to collect both forward and side scattered light from the cells. Side scatter (SSC) refers to an orthogonal, or at least transverse, alignment of the optical axes of the excitation and collection optics. Forward scatter (FSC) refers to a transmission mode optical set up. A typical machine will have one detector arranged for forward scatter collection and several detectors arranged for side scatter collection. Usually fluorescence data at different emission wavelengths are collected by multiple detectors arranged in side scatter. As well as for cell counting, the instrument is sensitive to a variety of cell properties, such as size, morphology, membrane roughness, granularity of the cytoplasm and many others. Generally each cell type has a unique combination of measured properties, including fluorescence, and correlation of FSC and SSC signals, which allow the cell type of each cell to be identified. Moreover, healthy and diseased cells of the same type may be distinguishable.
Before using a flow cytometer to measure fluorescence signals from cell samples marked with fluorescent tags, it is first necessary to calibrate the system so that the absolute intensity of the fluorescence signal measured by each combination of excitation source and detector is known. Otherwise, for example, test results from different instruments cannot be compared and results from one day to the next cannot be compared.
One necessary aspect of calibration of the instrument is to quantify the amount of fluorescence that is measured at each emission band or channel of interest. Each excitation/emission combination can thus be ascribed a calibration value with which measured data needs to be scaled. These calibration values can be represented in a calibration curve as a function of emission wavelength, for example. Now, assuming the source, detector, intervening optics, and other parameters are not changed, the signals obtained from a subsequent sample can have their fluorescence intensities quantified by scaling the measured values with the appropriate calibration values. The number of fluorescence molecules per cell, or whatever other parameter is being measured, can then be reliably calculated.
In this respect it is noted that the detectors used are typically photomultiplier tubes (PMTs) and these are well known for having sensitivity that changes over time, in particular from session to session. This is inherent in their design being essentially high voltage biased vacuum tubes operating at or near breakdown with avalanching effects. However, PMTs are difficult to displace with other detectors in view of their extremely high sensitivity.
A separate PMT may be used for each wavelength channel. On the other hand, it is also known to use multichannel PMTs where each channel receives a different wavelength band. The company Hamamatsu markets such a multichannel PMT under product numbers H9530 and H9797 series. This product integrates an 8-channel PMT with an input side optical arrangement of dichroic mirrors to spectrally sort an input beam into the 8-channels. A full technical description of this product is provided in EP 1 666 857 A1 [1] as well as in Hamamatsu product literature which refers to the suitability of this product for flow cytometers, cell sorters, cell analyzers, laser scanning microscopy and other specified uses.
For these reasons calibration is a major concern when using a flow cytometer. The typical approaches are to perform calibration runs with calibration beads and/or to run test experiments with standard samples of known and reproducible fluorescence properties.
FIG. 1 shows schematically the flow cell region of a conventional flow cytometer using multiple PMTs. The flow cytometer has a flow cell 10 which receives a sample inlet tube 12. The sample inlet tube 12 is connected to an inner capillary tube 14 of the flow cell 10 which is radially enclosed prior to its termination by a sheath 16 which has a sheath inlet 18 connected to a sheath fluid inlet tube (not shown). As considered in the flow direction, the sheath 16 reduces in its cross-sectional diameter and the inner capillary tube 14 terminates leaving the sample fluid and sheath fluid flowing together along a capillary tube 20. After this termination, the sample flows radially confined to the central region of the flow by virtue of laminar flow at the interface between the sample fluid and the sheath fluid. The aim of this sheath arrangement is to allow good optical access to the sample in a flow tube that is sufficiently large in diameter to avoid blockages.
The various optical components for excitation and collection are arranged about a measurement region of the capillary tube 20. A laser 22 outputs a laser beam 24 that is focused by a lens 26 on the central region of the capillary tube 20 so as to intersect with the sample. Fluorescence from the sample excited by the laser 22 is then collected through a collection lens 28 and spectral sorting arrangement 30, comprising mirrors 32 and filters 34, which divides the fluorescence into different wavelength bands. Each color component is directed to a suitable PMT 361, 362, 363, 364 as illustrated with the example of four PMTs. A FSC detector 35 and SSC detector 37 may also be provided and are schematically depicted. Moreover, although not illustrated, multiple lasers may be provided to cover all excitation wavelengths of interest. Also, the laser or lasers may be tuneable.
FIG. 2 is a graph showing schematically aspects of a conventional calibration process for an 8-channel detector assembly using 8 PMTs. The channels cover respective wavelength bands centered at λ1, λ2, λ3, λ4, λ5, λ6, λ7, λ8, wherein these wavelengths will generally be unevenly spaced and centered on a particular emission band of interest. A standard calibration method is now described. A set of broadband fluorescent calibration beads is supplied to the instrument to acquire calibration data on each of the 8 channels. The respective measured intensities for the channels are illustrated by the crosses in the figure and have values Ic(λi) where i=1 to 8. Optionally some blank beads may also be run to obtain a zero baseline intensity I0. In the illustrated example, no baseline is shown. The calibration beads have a known spectral response fB(λ) as plotted in the figure. In the illustrated example, it can thus be seen that generally the measured intensities λ1 to λ7 are lower than they should be, but λ8 is approximately correct. Each of the channels is then normalized according to the strength of the measured signal taking account of the calibration bead response, namely the normalization factor Ni for each channel is given by the equation:
      N    i    =      c    ·                            f          B                ⁡                  (                      λ            i                    )                                                  I            C                    ⁡                      (                          λ              i                        )                          -                  I          0                    where c is an arbitrary constant. The normalization factors are then used in subsequent measurements to adjust the measured intensities IM according to the formula, so that a processed intensity IP is arrived at by the formula IP=Ni·IM. The measured intensities of samples of interest are thus adjusted to take account of the measured intensities for the calibration beads.
Each wavelength channel is thus normalized according to its sensitivity. This information is typically stored in software. This is a standard approach along similar principles to what is discussed throughout the literature on flow cytometry, for example see the references [2, 3, 4].